Hydroxyl radical protein footprinting coupled with mass spectrometry is a powerful tool used to probe protein structure, interactions, and conformational changes in the solution phase. A hydroxyl radical dosimeter is used to ensure that different proteins oxidized under the same amount of radical. However, AlexaFluor is not an ideal dosimeter for hydroxyl radicals: it is a loss-of-signal dosimeter which lowers its sensitivity, and has several oxidation products, each with different properties. In biological research, previous reports have proposed terephthalic acid (TPA) as a hydroxyl radical dosimeter. Unlike AlexaFluor, TPA gains fluorescence upon oxidation and has only one oxidation product upon reaction with hydroxyl radicals. Our study investigated the suitability of TPA as a hydroxyl radical dosimeter for the normalization of hydroxyl radical protein footprinting data. However, further data indicates that TPA maybe not an ideal radical dosimeter to use in hydroxyl radical protein footprinting due to its complex relationship between radical dose and fluorescence. An alternative approach using tyrosine amide as both a scavenger and a radical dosimeter coupled with mass spectrometry to measure the oxidization level of tyrosine amide shows promise for normalization of footprinting data. Initial data indicates that tyrosine amide acts as a suitable replacement for glutamine as a scavenger and provides a dosimetry response that appears to be linearly related to effective hydroxyl radical dose.
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